TMS-EDTA

Figure 13.8 TMS-EDTA can be used to modify an inorganic substrate to containing EDTA chelating groups for complexation with metal ions.

In 10 mM acetate buffer at pH 5.5 and with 4 /tM EDTA present to reduce the catal5dic effects of iron salts, the order of inhibition was determined to be CN- > Ng- > F- > I- > NO3- > Cl- > Br- > OCN" > SCN- > SeCN- > (CIO4-, tetraborate, boric acid, phosphate, sulfate and cacodylate). Clearly the strongest inhibitors are those with metal binding capabilities although this features alone does not readily correlate the series. Indeed, there are complexities which are apparent imder detailed scrutiny and which suggest a very compUcated pattern of inhibitory action by these anions. Curzon and his co-workers and other investigators have carried out detailed studies of the inhibition by azide, cyanide, the halides, and mixtures of various anionic inhibitors. 

Although acetic acid accelerates the rate of dissociation of [Eu(cydta)] and [Tm(edta)] ions, mixed acetato-complexes are not involved in the reactions. ... 

Oxidation specifically attacks thymine (57,58) and is applicable to all nucleic acid analytes. This strategy aims to either eliminate or alter thymine and thymine like-moieties for better analyte recognition. Oxidation with osmium tetroxide (5% OSO4) occurs at 300 pg/ml, with nucleic acids or DNA (plasmid) in 100 mM NaCl, 10 mM Tris (pH 7.6) and 1 mM EDTA. Incubation is at 70 C for 90 minutes. Standard ethanol extraction follows, with resuspension in tm-EDTA buffer. A series of chromatograms, including thymine altered by ozone and osmium are in press. 

Liu Y, TM Louie, J Payne, J Bohuslavek, H Bolton, L Xun (2001) Identification, purification, and characterization of iminodiacetate oxidase from the EDTA-degrading bacterium BNC-1. Appl Environ Microbiol 67 696-701. 

Figure 6. Scanning electron micrograph (SEM) of n-GaAs surface electrochemically etched with a scanning electrochemical and tunneling microscope (SETM). Etching was accomplished in Aq. 5 mU NaOH, 1 mM EDTA. Photoelectric current - 0.7 /iA, Scan rate - 0.1 /tm/sec, bias voltage — 4 V. Tip was moved in an "L" pattern. Reproduced with permission of Ref. 89. Copyright 1987 The Electrochemical Society Inc.

List of Abbreviations PCR, polymerase chain reaction RT-PCR, reverse transcription polymerase chain reaction DNA, deoxyribonucleic acid RNA, ribonucleic acid RNase, ribonuclease mRNA, messenger RNA GABAa, y-aminobutyric acid type A cRNA, copy RNA dNTPs, deoxy nucleoside triphosphates MMLV, Mouse Moloney murine leukemia vims RT, reverse transcriptase bp, base pair Tm, melting temperature DEPC, diethylpyrocarbonate OD, optical density mL, milliliter SA-PMPs, streptavidin paramagnetic particles dT, deoxy thymidine DTT, dithiothreitol DNase, deoxyribonuclease RNasin, ribonuclease inhibitor UV, ultraviolet TBE, Tris-borate, 1 mM EDTA EDTA, ethylenediaminetetraacetic acid Buffer RET, guanidium thiocyanate lysis buffer PBS, phosphate buffered saline NT2, Ntera 2 neural progenitor cells... 

Reductase Activity Measurement. Reductase activity was measured by the reduction of 200 /iM veratraldehyde to veratryl alcohol in the presence of 250 /tM NADPH. The reaction was carried out in 20 raM Tris buffer pH 7.4 (optimal pH = 6) with 20% glycerol and 1 mM EDTA. The decrease in absorbance of NADPH at 365 nm was measured. NADPH was slowly oxidized in those mixtures which were not purified by ion exchange even in the absence of veratraldehyde. This unspecific reaction was considered in the calculation of the reductase activity. One unit of reductase reduced 1 /imol min-1 of veratraldehyde to veratryl alcohol at room temperature (25°C). 

The blood samples are mixed with K-EDTA (1 mg/ml blood) or heparin (5 IE/ml heparin sodium) to prevent clotting. The blood is centrifuged at 3000 rpm for 7 min. The plasma and the buffy coat are removed and discarded. The packed erythrocytes are resuspended in hitrated HEPES-buffer containing 0.25 % human albumin and the haematocrit value is fixed to < 1 %. The red blood cells are altered by one or several stress factors mentioned above. A sample of 2 ml of the stressed suspension is applied to the measuring device and the passage time of a population of 250 erythrocytes (tm) is determined. Cells remaining in the pore for more than 100 ms (tm > 100 ms) lead to a rheological occlusion. 

The substrate and two products are separated on a //.Bondapak TM C]8 column (3.9 mm x 300 mm). The mobile phase consisted of 0.05 M acetate buffer (pH 4.7), 10 mM Na2S205, 1 mM EDTA, and 25% methanol. The column effluent was monitored by fluorimetry with exdtation, and emission wavelengths being 295 and above 340 nm, respectively. 

The substrates and products just noted were separated on an Ultrasphere I.P. CI8 column (4.6 mm x 250 mm, 5 /tm). The mobile phase contained 75 mAf sodium phosphate (pH 2.75), 1 mM sodium octylsulfate, 500 fiM EDTA, and 13% (v/v) acetonitrile. Quantitation was by electrochemical detection of the products using 0.75 V versus an Ag/AgCl reference electrode. 

As mentioned in Sec. I.B. the efficiency of separation is measured by the HETP, so it is important to study the influence of increasing concentrations of EDTA on the HETP. The effect of this variable on HETP was monitored by using the separation of Tm-Er as a suitably representative system. The results obtained are listed in Table 9. 

The sample described in Table 10 has been separated by employing the following conditions dictated by the results of earlier fundamental studies. The five-column system was employed for the separation. Neutral salt added to 0.075 mol/L EDTA at a pH value of 7.3 was used as the ion displacer. The column ratio was 1 2. The temperature of the process was fixed at 75°C. All products exited from the last column. Yb-(169, 175) was used to define the boundary curves of Lu-Yb and Tm-Yb, while Tb-160 was used to define the boundary curves of Tb-DY and Tb-Gd. In addition, Tm-170 and Gd-(153, 159) were used to determine the displacement curves of Tm and Gd. Other element boundaries were defined by using spectrophotometry. The results obtained are shown in Fig. 9. 

Fig. 2 Multiplex PCR profile of exons in Duchenne or Becker muscular dystrophy genes combined with two flanking standards. Capillary 40 cm (65 cm total length) X 75 tm polyacrylamide coated background electrolyte 0.5% poly(ethylene oxide), 1 MDa in IX Tris-borate-EDTA, lO tM aminoacridine, 2 nM Vistra Green field strength 108 V/cm detection LIF, 488 nm. [Reprinted with permission from/. Chromatogr. A 781 295 (1997), copyright 1997, Elsevier Science Publishers.]...

Table 13. PNA duplex characteristics I PNA/RNA and PNA/DNA duplexes show increased stability when compared with natural duplexes (Conditions 10 mM phosphate buffer 0.1 mM EDTA 100 mM NaCl pH 7.0). II Dependence of Tm-values of PNA/DNA and natural duplexes on counterion strength. From [223]...

EDTA can effectively separate most of the rare earths from each other. Only for the pairs Eu-Gd, Dy-Y, and Yb-Lu does it not work so well. Separation of Dy-Y with HEDTA is, however, possible. HEDTA also shows good results for Tm-Yb-Lu, and for La-Ce-Pr-Nd-Y-Sm, and Ho-Er-Tm-Yb-Lu mixmres. 

TM siudiei of ihc conoskm of carbon stcci in an ammontaied EDTA solution [70J. (With ihc permission of John Wiley and Son>, Chichester.)... 

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